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EP4211244A1 - Zusammensetzungen und verfahren zur cd38-modifizierung - Google Patents

Zusammensetzungen und verfahren zur cd38-modifizierung

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Publication number
EP4211244A1
EP4211244A1 EP21787233.2A EP21787233A EP4211244A1 EP 4211244 A1 EP4211244 A1 EP 4211244A1 EP 21787233 A EP21787233 A EP 21787233A EP 4211244 A1 EP4211244 A1 EP 4211244A1
Authority
EP
European Patent Office
Prior art keywords
cell
grna
cells
domain
nucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21787233.2A
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English (en)
French (fr)
Inventor
John LYDEARD
Mark Jones
Michael PETTIGLIO
Tirtha Chakraborty
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vor Biopharma Inc
Original Assignee
Vor Biopharma Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vor Biopharma Inc filed Critical Vor Biopharma Inc
Publication of EP4211244A1 publication Critical patent/EP4211244A1/de
Pending legal-status Critical Current

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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A61K39/4611—
    • A61K39/4622—
    • A61K39/4631—
    • A61K39/464411—
    • A61K39/464426—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/02—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2) hydrolysing N-glycosyl compounds (3.2.2)
    • C12Y302/02006—NAD(P)+ nucleosidase (3.2.2.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/17—Hinge-spacer domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/21—Transmembrane domain
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
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    • C12N2510/00—Genetically modified cells

Definitions

  • the therapy can deplete not only the pathological cells intended to be targeted, but also non-pathological cells that may express the targeted antigen.
  • This "on-target, off-disease" effect has been reported for some CAR-T therapeutics, e.g., those targeting CD19 or CD33. If the targeted antigen is expressed on the surface of cells required for survival or the subject, or on the surface of cells the depletion of which is of significant detriment to the health of the subject, the subject may not be able to receive the immunotherapy, or may have to face severe side effects once administered such a therapy.
  • an immunotherapy targeting an antigen that is expressed on the immune effector cells that constitute the immunotherapy, e.g., on the surface of CAR-T cells, which may result in fratricide and render the respective therapeutics ineffective or virtually impossible to produce.
  • compositions, methods, strategies, and treatment modalities that address the detrimental on-target, off-disease effects of certain immunotherapeutic approaches, e.g., of immunotherapeutic s comprising lymphocyte effector cells targeting a specific antigen in a subject in need thereof, such a s CAR-T cells or CAR- NK cells.
  • gRNA guide RNAs
  • the gRNA comprises a targeting domain, wherein the targeting domain comprises a sequence of any one of SEQ ID NOs: 12, 58-84, 85-155, and 180-190.
  • the gRNA comprises a first complementarity domain, a linking domain, a second complementarity domain which is complementary to the first complementarity domain, and a proximal domain.
  • the gRNA is a single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • the gRNA comprises one or more nucleotide residues that are chemically modified.
  • the gRNA comprises one or more nucleotide residues that comprise a 2'O-methyl moiety. In some embodiments, the gRNA comprises one or more nucleotide residues that comprise a phosphorothioate. In some embodiments, the gRNA comprises one or more nucleotide residues that comprise a thioPACE moiety.
  • aspects of the present disclosure provide methods of producing a genetically engineered cell, comprising providing a cell, and contacting the cell with (i) any of the gRNAs described herein, a gRNA targeting a targeting domain targeted by any of the gRNAs described herein; and (ii) an RNA-guided nuclease that binds the gRNA, thus forming a ribonucleoprotein (RNP) complex under conditions suitable for the gRNA of (i) to form and/or maintain an RNP complex with the RNA-guided nuclease of (ii) and for the RNP complex to bind a target domain in the genome of the cell.
  • RNP ribonucleoprotein
  • the contacting comprises introducing (i) and (ii) into the cell in the form of a pre-formed ribonucleoprotein (RNP) complex. In some embodiments, the contacting comprises introducing (i) and/or (ii) into the cell in the form of a nucleic acid encoding the gRNA of (i) and/or the RNA-guided nuclease of (ii). In some embodiments, the nucleic acid encoding the gRNA of (i) and/or the RNA-guided nuclease of (ii) is an RNA, preferably an mRNA or an mRNA analog. In some embodiments, the ribonucleoprotein complex is introduced into the cell via electroporation.
  • the RNA-guided nuclease is a CRISPR/Cas nuclease.
  • the CRISPR/Cas nuclease is a Cas9 nuclease.
  • the CRISPR/Cas nuclease is an spCas nuclease.
  • the CRISPR/Cas nuclease is a Cpf1 nuclease.
  • the cell is a hematopoietic cell.
  • the cell is a hematopoietic stem cell. In some embodiments, the cell is a hematopoietic progenitor cell. In some embodiments, the cell is an immune effector cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T-lymphocyte. [0009] Aspects of the present disclosure provide genetically engineered cells obtained by any of the methods described herein. Aspects of the present disclosure provide cell populations comprising the genetically engineered cells described herein.
  • aspects of the present disclosure provide cell populations comprising a genetically engineered cell, wherein the genetically engineered cell comprises a genomic modification that consists of an insertion or deletion immediately proximal to a site cut by an RNA-guided nuclease when bound to a gRNA comprising a targeting domain as described in any of Tables 1-5.
  • the genomic modification is an insertion or deletion generated by a non-homologous end joining (NHEJ) event.
  • NHEJ non-homologous end joining
  • the genomic modification is an insertion or deletion generated by a homology-directed repair (HDR) event.
  • the genomic modification results in a loss-of function of CD38 in a genetically engineered cell harboring such a genomic modification. In some embodiments, the genomic modification results in a reduction of expression of CD38 to less than 25%, less than 20% less than 10% less than 5% less than 2% less than 1%, less than 0.1%, less than 0.01%, or less than 0.001% as compared to the expression level of CD38 in wild-type cells of the same cell type that do not harbor a genomic modification of CD38. In some embodiments, the genetically engineered cell is a hematopoietic stem or progenitor cell.
  • the genetically engineered cell is an immune effector cell.
  • the genetically engineered cell is a T-lymphocyte.
  • the immune effector cell expresses a chimeric antigen receptor (CAR).
  • the CAR targets CD38.
  • the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient and to generate differentiated progeny of all blood lineage cell types in the recipient.
  • the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 50%.
  • the cell population is characterized by the ability to engraft CD38- edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 60%.
  • the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 70%. In some embodiments, the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 80%. In some embodiments, the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 90%. In some embodiments, the cell population CD38-edited hematopoietic stem cells that are characterized by a differentiation potential that is equivalent to the differentiation potential of non-edited hematopoietic stem cells.
  • aspects of the present disclosure provide methods comprising administering to a subject in need thereof any of the genetically engineered cells described herein or any of the cell populations described herein.
  • the subject has or has been diagnosed with a hematopoietic malignancy.
  • the method further comprises administering to the subject an effective amount of an agent that targets CD38, wherein the agent comprises an antigen-binding fragment that binds CD38.
  • FIGURE 1 depicts the crystal structure of CD38, including the conformational closure of the catalytic site of human CD38 induced by calcium (retrieved from the RCSB Protein Data Bank www.rcsb.org/structure/3F6Y).
  • FIGURE 2 is a schematic showing the location of the guide RNAs described herein relative to the human CD38 gene.
  • FIGURES 3A-3F are graphs depicting the INDEL (insertion/deletion) distribution for human T lymphoblast MOLT-4 cells edited with the indicated exemplary gRNAs.
  • FIGURE 3A shows editing with gRNA CD38-23, which resulted in a total efficiency of 78.7%.
  • FIGURE 3B shows editing with gRNA CD38-7, which resulted in a total efficiency of 82.8%.
  • FIGURE 3C shows editing with gRNA CD38-12 which resulted in a total efficiency of 80.4%.
  • FIGURE 3D shows editing with gRNA CD38-26, which resulted in a total efficiency of 76.3%.
  • FIGURE 3E shows editing with gRNA CD38-29, which resulted in a total efficiency of 88.6%.
  • FIGURE 3F shows editing with gRNA CD38- 9, which resulted in a total efficiency of 85.3%.
  • the X-axis indicates the size of the INDEL and the Y-axis indicates the percentage of the specific INDEL in the mixture.
  • FIGURES 4A-4B show CD38-modified Molt-4 cells.
  • the expression of CD38 was assessed by flow cytometry.
  • FIGURE 4A shows CD38 expression in control cells edited with a control guide (does not target CD38), or cells edited with the indicated CD38 gRNAs, from top to bottom: gRNA CD38-23, gRNA CD38-24, gRNA CD38-25, gRNA CD38-26, gRNA CD38-27, gRNA CD38-29, and gRNA CD38-3O.
  • the percentage CD38+ cells is shown in the right panel, and the percentage CD38- cells is shown in the left panel.
  • the X-axis indicates the intensity of antibody staining and the Y-axis corresponds to the cell number.
  • FIGURE 4B shows CD38 expression, from top to bottom, in live/dead cells, mock electroporated cells ("Mock”), unstained control cells, wildtype Molt-4 cells, and cells edited with a guide control (scrambled, non-targeting guide, "Guide Control”).
  • FIGURES 5A-5G are graphs depicting the INDEL (insertion/deletion) distribution for human T lymphoblast MOLT-4-cells edited with the indicated exemplary gRNA.
  • FIGURE 5A shows editing with gRNA CD38-23, which resulted in a total efficiency of 85.8%.
  • FIGURE 5B shows editing with gRNA CD38-12, which resulted in a total efficiency of 66.8%.
  • FIGURE 5C shows editing with gRNA CD38-7, which resulted in a total efficiency of 64.2%.
  • FIGURE 5D shows editing with gRNA CD38-7, which resulted in a total efficiency of 70.8%.
  • FIGURE 5E shows editing with gRNA CD38-7, which resulted in a total efficiency of 74.4%.
  • FIGURE 5F shows editing with gRNA CD38-7, which resulted in a total efficiency of 84%.
  • FIGURE 5G shows editing with gRNA CD38-7, which resulted in a total efficiency of 80.4%.
  • the X-axis indicates the size of the INDEL, and the Y-axis indicates the percentage of the specific INDEL in the mixture.
  • FIGURE 6 is a diagram showing the crystal structure of the extracellular domain (ECD) of CD38, marking the locations of cysteine 296 and tryptophan 46 (left), and showing the predicted overall CD38 structure (right).
  • the crystal structure file can be found at www.rcsb.org/3d-view/lYH3.
  • the predicted structure can be found at alphafold . ebi . ac.uk/entry/P28907.
  • FIGURES 7A-7C are graphs showing loss of CD38 surface expression on CD37+ cells 2 days and 5 days following editing with the indicated CD38 gRNA.
  • FIGURE 7A shows the percentage of cells positive for CD38 on their surfaces.
  • FIGURE 7B shows the CD38 geometric mean fluorescence intensity (gMFI).
  • FIGURE 7C shows the percentage of mock (the gMFI of CD38-edited cells relative to the gMFI of mock electroporated cells multiplied by 100. Each symbol represents cells from a different donor. For the columns in FIGURES 7B and 7C, the symbols correspond, from left to right, to Mock, gRNA CD38-8, gRNA CD38-11, and gRNA CD38-7.
  • FIGURES 8A and 8B are graphs showing CD38 editing efficiency and an INDEL spectrum in CD34+ hematopoietic stem and progenitor cells (HSPCs).
  • FIGURE 8A shows the percent editing efficiency for CD34+ obtained from three different human donors and electroporated with the indicated CD38 gRNA.
  • FIGURE 8B shows an INDEL spectrum at 5 days post-electroporation.
  • FIGURE 9 is a schematic showing the location of the guide RNAs described herein relative to the human CD38 gene.
  • the lower, shaded box denotes the position of exon 1 within the CD38 gene.
  • Arrows denote the positions targeted by gRNAs selected for examination in Examples 6-8.
  • FIGURES 10A and 10B are graphs showing CD38 editing efficiency and CD38 surface expression in CD34+ hematopoietic stem and progenitor cells (HSPCs) at various days post electroporation with the indicated CD38 gRNAs.
  • FIGURE 10A shows the of percentage CD38 editing efficiency in CD34+ hematopoietic stem and progenitor cells (HSPCs) positive cells.
  • FIGURE 10B shows the percentage of CD38 positive cells.
  • FIGURES 11A and 11B are graphs showing total THP-1 cells and viability at various days post electroporation with the indicated CD38 gRNAs, a control gRNA (gCtrl), a CD33 gRNA (gCD33), mock electroporated (Mock), or wild-type cells.
  • FIGURE 11A shows the total cell number.
  • FIGURE 11B shows the percent sample viability .
  • FIGURES 12A-12C are graphs showing CD38 editing efficiency and loss of expression of CD38 in THP-1 cells at various days post electroporation with the indicated CD38 gRNAs or a control gRNA (Control).
  • FIGURE 12A shows the percentage CD38 editing efficiency.
  • FIGURE 12B shows CD38 RNA transcript expression level as a percentage of control.
  • FIGURE 12C shows the percentage of cells positive for CD38 surface expression.
  • FIGURES 13A-13C are graphs showing colony counts for CD38-edited CD34+ hematopoietic stem and progenitor cells (HSPCs) electroporated with the indicated CD38 gRNA or mock electroporated (Mock), as measured using a STEMvisionTM colony counting assay.
  • FIGURE 13A shows erythroid (BFU-E: burst forming unit) colony formation.
  • FIGURE 13B shows multipotential myeloid progenitor cell (GEMM: colony forming units of multipotential myeloid progenitor cells) colony formation.
  • FIGURE 13C shows granulocyte/macrophage (G/M/GM: granulocyte/macrophage) colony formation. 400 CD34+ HSPCs for each sample in duplicate.
  • FIGURES 14A-14C are graphs showing the INDEL spectra produced by CRISPR editing of human donor hematopoietic stem and progenitor cells (HSPCs) using the indicated CD38 gRNAs.
  • HSPCs human donor hematopoietic stem and progenitor cells
  • FIGURE 14A shows editing with gRNA CD38-8.
  • FIGURE 14B shows editing with gRNA CD38-11.
  • FIGURE 14C shows editing with gRNA CD38-7.
  • compositions, methods, strategies, and treatment modalities related to genetically modified cells e.g., hematopoietic cells, that are deficient in the expression of an antigen targeted by a therapeutic agent, e.g., an immunotherapeutic agent.
  • a therapeutic agent e.g., an immunotherapeutic agent.
  • the genetically modified cells provided herein are useful, for example, to mitigate, or avoid altogether, certain undesired effects, for example, any on- target, off-disease cytotoxicity, associated with certain immunotherapeutic agents.
  • Such undesired effects associated with certain immunotherapeutic agents may occur, for example, when healthy cells within a subject in need of an immunotherapeutic intervention express an antigen targeted by an immunotherapeutic agent.
  • a subject may be diagnosed with a malignancy associated with an elevated level of expression of a specific antigen, which is not typically expressed in healthy cells, but may be expressed at relatively low levels in a subset of non-malignant cells within the subject.
  • Administration of an immunotherapeutic agent e.g., a CAR-T cell therapeutic or a therapeutic antibody or antibody-drug-conjugate (ADC) targeting the antigen, to the subject may result in efficient killing of the malignant cells, but may also result in ablation of non-malignant cells expressing the antigen in the subject.
  • ADC antibody-drug-conjugate
  • compositions, methods, strategies, and treatment modalities provided herein address the problem of on-target, off-disease cytotoxicity of certain immunotherapeutic agents.
  • some aspects of this disclosure provide genetically engineered cells harboring a modification in their genome that results in a lack of expression of an antigen, or a specific form of that antigen, targeted by an immunotherapeutic agent.
  • Such genetically engineered cells, and their progeny are not targeted by the immunotherapeutic agent, and thus not subject to any cytotoxicity effected by the immunotherapeutic agent.
  • Such cells can be administered to a subject receiving an immunotherapeutic agent targeting the antigen, e.g., in order to replace healthy cells that may have been targeted and killed by the cytotherapeutic agent, and/or in order to provide a population of cells that is resistant to targeting by the cytotherapeutic agent.
  • an immunotherapeutic agent targeting the antigen e.g., in order to replace healthy cells that may have been targeted and killed by the cytotherapeutic agent, and/or in order to provide a population of cells that is resistant to targeting by the cytotherapeutic agent.
  • genetically engineered hematopoietic cells provided herein, e.g., genetically engineered hematopoietic stem or progenitor cells, may be administered to the subject that do not express the antigen, and thus are not targeted by the cytotherapeutic agent.
  • Such hematopoietic stem or progenitor cells are able to re-populate the hematopoietic niche in the subject and their progeny can reconstitute the various hematopoietic lineages, including any that may have been ablated by the cytotherapeutic agent.
  • CD38 also referred to as cyclic ADP ribose hydrolase
  • CD38 is a 45 KDa glycoprotein that synthesizes the second messages cyclic ADP-ribose and nicotinate-adenine dinucleotides phosphate
  • CD38 has also been reported to have cyclic adenosine 5'- diphosphate ribose (cADPr) hydrolase activity and functions as a receptor on immune cells.
  • cADPr cyclic adenosine 5'- diphosphate ribose
  • CD38 is naturally present in two opposite membrane orientations. See, e.g., Liu et al. PNAS (2017) 114(31: 8283-8288. The majority of CD38 has a type II membrane orientation, with the catalytic site facing the outside of the cell.
  • CD38 can also localize to the inner surface of cell membranes, such as nuclear membrane, mitochondria membrane, and endoplasmic reticulum.
  • a small fraction of CD38 is a type III plasma membrane protein with the catalytic site directed intracellularly. Soluble intra- and extracellular forms of CD38 have also been described.
  • CD38 The gene encoding CD38 consists of 8 exons with the protein being reported to be present in two isoforms, based on analysis using the Genome Aggregation Database (gnomAD).
  • CD38 is typically expressed on the surface of healthy plasma cells and other lymphoid and myeloid cells, e.g., B-cells, NK cells, myeloid precursors, and activated T and B lymphocytes, erythrocytes, platelets, progenitor cells, including cord blood cells. See, e.g., Morandi et al. Front. Immunol. (2016).
  • lymphoid and myeloid cells may also be expressed in solid tissues, such as the intestinal epithelial cells, lamina basement, epithelial cells in the prostate, cells of the central nervous system, beta cells of the pancreas, as well as retina and muscle cells.
  • CD38 In addition to its normal expression on healthy cells, CD38 is also highly expressed on the surface of hematologic cancer cells. For example, high and uniform CD38 expression has been reported on malignant plasma cells, such as multiple myeloma cells. CD38 is also utilized as a prognostic marker in leukemia, such as B-cell chronic lymphocytic leukemia (B-CLL). Due to the high level of expression on such malignant cells, CD38 is an attractive target for immunotherapies for such indications, for which numerous therapeutics have been developed.
  • B-CLL B-cell chronic lymphocytic leukemia
  • CAR T cells CD38-specific chimeric antigen receptors
  • antibody therapeutics e.g., daratumumab (Darzalex, Janssen Pharmaceuticals), isatuximab (SAR650984, Sanofi), MOR202 (MorphoSys, I-Mab Biopharma), TAK-079 (Takeda).
  • CD38-specific CAR T cell therapy is associated with fratricide of the CAR T cells, reducing efficacy of the therapy. See, e.g., Huang et al. J Zhejiang Univ Sci B. 2020 Jan; 21(1): 29-41.
  • gRNAs that have been developed to specifically direct genetic modification of the gene encoding CD38. Also provided herein is use of such gRNAs to produce genetically modified cells, such as hematopoietic cells, immune cells, lymphocytes, and populations of such cells, that are deficient in CD38 or have reduced expression of CD38 such that the modified cells are not recognized by CD38-specific immunotherapies. Also provided herein are methods involving administering such cells, or compositions thereof, to subjects to address the problem of on-target, off-disease cytotoxicity of certain immunotherapeutic agents.
  • the genetically modified cells are hematopoietic cells that are deficient in CD38 or have reduced expression of CD38 that are capable, for example, of developing into lineage-committed cells, such as T cells that are deficient in CD38 or have reduced expression of CD38, and therefore, are resistant to killing by CD38-specific immunotherapies.
  • the genetically modified cells are immune cells, such as CD38-specific CAR T cells that are deficient in in CD38 or have reduced expression of CD38,and therefore, are resistant to fratricide killing by other CD38-specific CAR T cells.
  • Some aspects of this disclosure provide genetically engineered cells comprising a modification in their genome that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • the modification in the genome of the cell is a mutation in a genomic sequence encoding CD38.
  • the term "mutation,” as used herein, refers to a change (e.g., an insertion, deletion, inversion, or substitution) in a nucleic acid sequence as compared to a reference sequence, e.g., the corresponding sequence of a cell not having such a mutation, or the corresponding wild-type nucleic acid sequence.
  • a mutation in a gene encoding CD38 results in a loss of expression of CD38 in a cell harboring the mutation.
  • a mutation in a gene encoding CD38 results in the expression of a variant form of CD38 that is not bound by an immunotherapeutic agent targeting CD38, or bound at a significantly lower level than the non-mutated CD38 form encoded by the gene.
  • a cell harboring a genomic mutation in the CD38 gene as provided herein is not bound by, or is bound at a significantly lower level by an immunotherapeutic agent that targets CD38, e.g., an anti-CD38 antibody or chimeric antigen receptor (CAR).
  • an immunotherapeutic agent that targets CD38 e.g., an anti-CD38 antibody or chimeric antigen receptor (CAR).
  • compositions and methods for generating the genetically engineered cells described herein e.g., genetically engineered cells comprising a modification in their genome that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • compositions and methods provided herein include, without limitation, suitable strategies and approaches for genetically engineering cells, e.g., by using RNA- guided nucleases, such as CRISPR/Cas nucleases, and suitable RNAs able to bind such RNA- guided nucleases and target them to a suitable target site within the genome of a cell to effect a genomic modification resulting in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • RNA- guided nucleases such as CRISPR/Cas nucleases
  • suitable RNAs able to bind such RNA- guided nucleases and target them to a suitable target site within the genome of a cell to effect a genomic modification resulting in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • a genetically engineered cell e.g., a genetically engineered hematopoietic cell, such as, for example, a genetically engineered hematopoietic stem or progenitor cell or a genetically engineered immune effector cell
  • a genetically engineered cell is generated via genome editing technology, which includes any technology capable of introducing targeted changes, also referred to as "edits," into the genome of a cell.
  • RNA editing comprising the use of a RNA-guided nuclease, e.g., a CRISPR/Cas nuclease, to introduce targeted single- or double-stranded DNA breaks in the genome of a cell, which trigger cellular repair mechanisms, such as, for example, nonhomologous end joining (NHEJ), microhomology-mediated end joining (MMEJ, also sometimes referred to as “alternative NHEJ” or “alt-NHEJ”), or homology-directed repair (HDR) that typically result in an altered nucleic acid sequence (e.g., via nucleotide or nucleotide sequence insertion, deletion, inversion, or substitution) at or immediately proximal to the site of the nuclease cut.
  • NHEJ nonhomologous end joining
  • MMEJ microhomology-mediated end joining
  • HDR homology-directed repair
  • a base editor e.g., a nuclease-impaired or partially nuclease- impaired RNA-guided CRISPR/Cas protein fused to a deaminase that targets and deaminates a specific nucleobase, e.g., a cytosine or adenosine nucleobase of a C or A nucleotide, which, via cellular mismatch repair mechanisms, results in
  • Yet another exemplary suitable genome editing technology includes "prime editing,” which includes the introduction of new genetic information, e.g., an altered nucleotide sequence, into a specifically targeted genomic site using a catalytically impaired or partially catalytically impaired RNA-guided nuclease, e.g., a CRISPR/Cas nuclease, fused to an engineered reverse transcriptase (RT) domain.
  • the Cas/RT fusion is targeted to a target site within the genome by a guide RNA that also comprises a nucleic acid sequence encoding the desired edit, and that can serve as a primer for the RT. See, e.g., Anzalone et al. Nature (2019) 576 (7785): 149-157.
  • RNA-guided nuclease typically features the use of a suitable RNA-guided nuclease, which, in some embodiments, e.g., for base editing or prime editing, may be catalytically impaired, or partially catalytically impaired.
  • suitable RNA- guided nucleases include CRISPR/Cas nucleases.
  • a suitable RNA-guided nuclease for use in the methods of genetically engineering cells provided herein is a Cas9 nuclease, e.g., an spCas9 or an saCas9 nuclease.
  • RNA-guided nuclease for use in the methods of genetically engineering cells provided herein is a Cas12 nuclease, e.g., a Cas12a nuclease.
  • exemplary suitable Cas12 nucleases include, without limitation, AsCas12a, FnCas12a, other Cas12a orthologs, and Cas12a derivatives, such as the MAD7 system (MAD7TM, Inscripta, Inc.), or the Alt-R Cas12a (Cpf1) Ultra nuclease (Alt-R® Cas12a Ultra; Integrated DNA Technologies, Inc.). See, e.g., Gill et al. LIPSCOMB 2017.
  • a genetically engineered cell e.g., a genetically engineered hematopoietic cell, such as, for example, a genetically engineered hematopoietic stem or progenitor cell or a genetically engineered immune effector cell
  • a genetically engineered cell is generated by targeting an RNA-guided nuclease, e.g., a CRISPR/Cas nuclease, such as, for example, a Cas9 nuclease or a Cas12a nuclease, to a suitable target site in the genome of the cell, under conditions suitable for the RNA-guided nuclease to bind the target site and cut the genomic DNA of the cell.
  • a CRISPR/Cas nuclease such as, for example, a Cas9 nuclease or a Cas12a nuclease
  • a suitable RNA-guided nuclease can be targeted to a specific target site within the genome by a suitable guide RNA (gRNA).
  • gRNA guide RNA
  • Suitable gRNAs for targeting CRISPR/Cas nucleases according to aspects of this disclosure are provided herein and exemplary suitable gRNAs are described in more detail elsewhere herein.
  • a CD38 gRNA described herein is complexed with a CRISPR/Cas nuclease, e.g., a Cas9 nuclease.
  • a CRISPR/Cas nuclease e.g., a Cas9 nuclease.
  • Cas9 nucleases are suitable for use with the gRNAs provided herein to effect genome editing according to aspects of this disclosure, e.g., to create a genomic modification in the CD38 gene.
  • the Cas nuclease and the gRNA are provided in a form and under conditions suitable for the formation of a Cas/gRNA complex, that targets a target site on the genome of the cell, e.g., a target site within the CD38 gene.
  • a Cas nuclease is used that exhibits a desired PAM specificity to target the Cas/gRNA complex to a desired target domain in the CD38 gene.
  • Suitable target domains and corresponding gRNA targeting domain sequences are provided herein.
  • a Cas/gRNA complex is formed, e.g., in vitro, and a target cell is contacted with the Cas/gRNA complex, e.g., via electroporation of the Cas/gRNA complex into the cell.
  • the cell is contacted with Cas protein and gRNA separately, and the Cas/gRNA complex is formed within the cell.
  • the cell is contacted with a nucleic acid, e.g., a DNA or RNA, encoding the Cas protein, and/or with a nucleic acid encoding the gRNA, or both.
  • genetically engineered cells as provided herein are generated using a suitable genome editing technology, wherein the genome editing technology is characterized by the use of a Cas9 nuclease.
  • the Cas9 molecule is of, or derived from, Streptococcus pyogenes (SpCas9), Staphylococcus aureus (SaCas9), or Streptococcus thermophilus (stCas9).
  • Cas9 molecules include those of, or derived from, Neisseria meningitidis (NmCas9), Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni (CjCas9), Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphth
  • catalytically impaired, or partially impaired, variants of such Cas9 nucleases may be used. Additional suitable Cas9 nucleases, and nuclease variants, will be apparent to those of skill in the art based on the present disclosure. The disclosure is not limited in this respect.
  • the Cas nuclease is a naturally occurring Cas molecule.
  • the Cas nuclease is an engineered, altered, or modified Cas molecule that differs, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule or a sequence of Table 50 of PCT Publication No. W02015/157070, which is herein incorporated by reference in its entirety.
  • a Cas nuclease is used that belongs to class 2 type V of Cas nucleases.
  • Class 2 type V Cas nucleases can be further categorized as type V-A, type V- B, type V-C, and type V-U. See, e.g., Stella et al. Nature Structural & Molecular Biology (2017).
  • the Cas nuclease is a type V-B Cas endonuclease, such as a C2cl. See, e.g., Shmakov et al. Mol Cell (2015) 60: 385-397.
  • the Cas nuclease used in the methods of genome editing provided herein is a type V-A Cas endonuclease, such as a Cpf1 (Cas12a) nuclease. See, e.g., Strohkendl et al. Mol. Cell (2016) 71: 1-9.
  • a Cas nuclease used in the methods of genome editing provided herein is a Cpf1 nuclease derived from Provetella spp. or Francisella spp., Acidaminococcus sp. (AsCpf1), Lachnospiraceae bacterium (LpCpf1), or Eubacterium rectale.
  • the Cas nuclease is MAD7TM (Inscripta).
  • CRISPR/Cas nucleases are suitable for use according to aspects of this disclosure.
  • dCas or nickase variants Cas variants having altered PAM specificities
  • Cas variants having improved nuclease activities are embraced by some embodiments of this disclosure.
  • a naturally occurring Cas9 nuclease typically comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which further comprises domains described, e.g., in PCT Publication No. W02015/157070, e.g., in Figs. 9A-9B therein (which application is incorporated herein by reference in its entirety).
  • the REC lobe comprises the arginine-rich bridge helix (BH), the RECI domain, and the REC2 domain.
  • the REC lobe appears to be a Cas9-specific functional domain.
  • the BH domain is a long alpha helix and arginine rich region and comprises amino acids 60-93 of the sequence of S. pyogenes Cas9.
  • the RECI domain is involved in recognition of the repeat: anti-repeat duplex, e.g., of a gRNA or a tracrRNA.
  • the RECI domain comprises two RECI motifs at amino acids 94 to 179 and 308 to 717 of the sequence of S. pyogenes Cas9.
  • the REC2 domain comprises amino acids 180-307 of the sequence of S. pyogenes Cas9.
  • the NUC lobe comprises the RuvC domain (also referred to herein as RuvC- like domain), the HNH domain (also referred to herein as HNH-like domain), and the PAM- interacting (PI) domain.
  • the RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves a single strand, e.g., the non-complementary strand of the target nucleic acid molecule.
  • the RuvC domain is assembled from the three split RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are often commonly referred to in the art as RuvCI domain, or N-terminal RuvC domain, RuvCII domain, and RuvCIII domain) at amino acids 1-59, 718-769, and 909-1098, respectively, of the sequence of S. pyogenes Cas9. Similar to the RECI domain, the three RuvC motifs are linearly separated by other domains in the primary structure, however in the tertiary structure, the three RuvC motifs assemble and form the RuvC domain.
  • the HNH domain shares structural similarity with HNH endonucleases, and cleaves a single strand, e.g., the complementary strand of the target nucleic acid molecule.
  • the HNH domain lies between the RuvC II- III motifs and comprises amino acids 775-908 of the sequence of S. pyogenes Cas9.
  • the PI domain interacts with the PAM of the target nucleic acid molecule and comprises amino acids 1099-1368 of the sequence of S. pyogenes Cas9.
  • Crystal structures have been determined for naturally occurring bacterial Cas9 nucleases (see, e.g., Jinek et al., Science, 343(6176): 1247997, 2014) and for S. pyogenes Cas9 with a guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) (Nishimasu et al., Cell (2014) 156:935-949; and Anders et al., Nature (2014) doi: 10.1038/naturel3579).
  • a guide RNA e.g., a synthetic fusion of crRNA and tracrRNA
  • a Cas9 molecule described herein exhibits nuclease activity that results in the introduction of a double strand DNA break in or directly proximal to a target site.
  • the Cas9 molecule has been modified to inactivate one of the catalytic residues of the endonuclease.
  • the Cas9 molecule is a nickase and produces a single stranded break. See, e.g., Dabrowska et al. Frontiers in Neuroscience (2016) 12(75). It has been shown that one or more mutations in the RuvC and HNH catalytic domains of the enzyme may improve Cas9 efficiency.
  • the Cas9 molecule is fused to a second domain, e.g., a domain that modifies DNA or chromatin, e.g., a deaminase or demethylase domain. In some such embodiments, the Cas9 molecule is modified to eliminate its endonuclease activity.
  • a Cas nuclease or a Cas/gRNA complex described herein is administered together with a template for homology directed repair (HDR).
  • HDR homology directed repair
  • a Cas nuclease or a Cas/gRNA complex described herein is administered without a HDR template.
  • a Cas9 nuclease is used that is modified to enhance specificity of the enzyme (e.g., reduce off-target effects, maintain robust on-target cleavage).
  • the Cas9 molecule is an enhanced specificity Cas9 variant (e.g., eSPCas9). See, e.g., Slaymaker et al. Science (2016) 351 (6268): 84-88.
  • the Cas9 molecule is a high fidelity Cas9 variant (e.g., SpCas9-HFl). See, e.g., Kleinstiver et al. Nature (2016) 529: 490-495.
  • Cas nucleases are known in the art and may be obtained from various sources and/or engineered/modified to modulate one or more activities or specificities of the enzymes.
  • PAM sequence preferences and specificities of suitable Cas nucleases e.g., suitable Cas9 nucleases, such as, for example, spCas9 and saCas9 are known in the art.
  • the Cas nuclease has been engineered/modified to recognize one or more PAM sequence.
  • the Cas nuclease has been engineered/modified to recognize one or more PAM sequence that is different than the PAM sequence the Cas nuclease recognizes without engineering/modification.
  • the Cas nuclease has been engineered/modified to reduce off-target activity of the enzyme.
  • a Cas nuclease is used that is modified further to alter the specificity of the endonuclease activity (e.g., reduce off-target cleavage, decrease the endonuclease activity or lifetime in cells, increase homology-directed recombination and reduce non-homologous end joining). See, e.g., Komor et al. Cell (2017) 168: 20-36.
  • a Cas nuclease is used that is modified to alter the PAM recognition or preference of the endonuclease.
  • SpCas9 recognizes the PAM sequence NGG, whereas some variants of SpCas9 comprising one or more modifications (e.g., VQR SpCas9, EQR SpCas9, VRER SpCas9) may recognize variant PAM sequences, e.g., NGA, NGAG, and/or NGCG.
  • SaCas9 recognizes the PAM sequence NNGRRT, whereas some variants of SaCas9 comprising one or more modifications (e.g., KKH SaCas9) may recognize the PAM sequence NNNRRT.
  • FnCas9 recognizes the PAM sequence NNG, whereas a variant of the FnCas9 comprises one or more modifications (e.g., RHA FnCas9) may recognize the PAM sequence YG.
  • the Cas 12a nuclease comprising substitution mutations S542R and K607R recognizes the PAM sequence TYCV.
  • a Cpf1 endonuclease comprising substitution mutations S542R, K607R, and N552R recognizes the PAM sequence TATV. See, e.g., Gao et al. Nat.
  • more than one (e.g., 2, 3, or more) Cas9 molecules are used.
  • at least one of the Cas9 molecule is a Cas9 enzyme.
  • at least one of the Cas molecules is a Cpf1 enzyme.
  • at least one of the Cas9 molecule is derived from Streptococcus pyogenes.
  • at least one of the Cas9 molecule is derived from Streptococcus pyogenes and at least one Cas9 molecule is derived from an organism that is not Streptococcus pyogenes.
  • a base editor is used to create a genomic modification resulting in a loss of expression of CD38, or in expression of a CD38 variant not targeted by an immunotherapy.
  • Base editors typically comprise a catalytically inactive or partially inactive Cas nuclease fused to a functional domain, e.g., a deaminase domain. See, e.g., Eid et al. Biochem. J. (2016) 475(11): 1955-1964; Rees et al. Nature Reviews Genetics (2016) 19:770-788.
  • a catalytically inactive Cas nuclease is referred to as "dead Cas" or "dCas.”
  • the endonuclease comprises a dCas fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA.
  • the endonuclease comprises a dCas fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation-induced cytidine deaminase (AID)).
  • the catalytically inactive Cas molecule has reduced activity and is, e.g., a nickase (referred to as "nCas").
  • the endonuclease comprises a dCas9 fused to one or more uracil glycosylase inhibitor (UGI) domains.
  • the endonuclease comprises a dCas9 fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA.
  • the endonuclease comprises a dCas9 fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation- induced cytidine deaminase (AID)).
  • the catalytically inactive Cas9 molecule has reduced activity and is nCas9.
  • the catalytically inactive Cas9 molecule (dCas9) is fused to one or more uracil glycosylase inhibitor (UGI) domains.
  • the Cas9 molecule comprises an inactive Cas9 molecule (dCas9) fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA.
  • ABE adenine base editor
  • the Cas9 molecule comprises a nCas9 fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA.
  • ABE adenine base editor
  • the Cas9 molecule comprises a dCas9 fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation-induced cytidine deaminase (AID)).
  • the Cas9 molecule comprises a nCas9 fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation-induced cytidine deaminase (AID)).
  • cytidine deaminase enzyme e.g., APOBEC deaminase, pmCDAl, activation-induced cytidine deaminase (AID)
  • Examples of suitable base editors include, without limitation, BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-CE3, VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)-BE3, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP.
  • Some aspects of this disclosure provide guide RNAs that are suitable to target an RNA-guided nuclease, e.g. as provided herein, to a suitable target site in the genome of a cell in order to effect a modification in the genome of the cell that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • guide RNA and "gRNA” are used interchangeably herein and refer to a nucleic acid, typically an RNA, that is bound by an RNA-guided nuclease and promotes the specific targeting or homing of the RNA-guided nuclease to a target nucleic acid, e.g., a target site within the genome of a cell.
  • a gRNA typically comprises at least two domains: a "binding domain,” also sometimes referred to as “gRNA scaffold” or “gRNA backbone” that mediates binding to an RNA-guided nuclease (also referred to as the "binding domain"), and a "targeting domain” that mediates the targeting of the gRNA-bound RNA- guided nuclease to a target site.
  • Some gRNAs comprise additional domains, e.g., complementarity domains, or stem- loop domains.
  • the structures and sequences of naturally occurring gRNA binding domains and engineered variants thereof are well known to those of skill in the art.
  • Some suitable gRNAs are unimolecular, comprising a single nucleic acid sequence, while other suitable gRNAs comprise two sequences (e.g., a crRNA and tracrRNA sequence).
  • Some exemplary suitable Cas9 gRNA scaffold sequences are provided herein, and additional suitable gRNA scaffold sequences will be apparent to the skilled artisan based on the present disclosure.
  • additional suitable scaffold sequences include, without limitation, those recited in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Publication No. WO2014/093694, and PCT Publication No. WO2013/176772.
  • the binding domains of naturally occurring spCas9 gRNA typically comprise two RNA molecules, the crRNA (partially) and the tracrRNA.
  • Variants of spCas9 gRNAs that comprise only a single RNA molecule including both crRNA and tracrRNA sequences, covalently bound to each other, e.g., via a tetraloop or via click- chemistry type covalent linkage, have been engineered and are commonly referred to as "single guide RNA" or "sgRNA.”
  • Suitable gRNAs for use with other Cas nucleases, for example, with Cas 12a nucleases typically comprise only a single RNA molecule, as the naturally occurring Cas 12a guide RNA comprises a single RNA molecule.
  • a suitable gRNA may thus be unimolecular (having a single RNA molecule), sometimes referred to herein as sgRNAs, or modular (comprising more than one, and typically two, separate RNA molecules).
  • sgRNAs single RNA molecule
  • modular comprising more than one, and typically two, separate RNA molecules.
  • a gRNA suitable for targeting a target site in the CD38 gene may comprise a number of domains.
  • a unimolecular sgRNA may comprise, from 5' to 3': a targeting domain corresponding to a target site sequence in the CD38 gene; a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain.
  • a gRNA as provided herein typically comprises a targeting domain that binds to a target site in the genome of a cell.
  • the target site is typically a double-stranded DNA sequence comprising the PAM sequence and, on the same strand as, and directly adjacent to, the PAM sequence, the target domain.
  • the targeting domain of the gRNA typically comprises an RNA sequence that corresponds to the target domain sequence in that it resembles the sequence of the target domain, sometimes with one or more mismatches, but typically comprises an RNA instead of a DNA sequence.
  • the targeting domain of the gRNA thus base-pairs (in full or partial complementarity) with the sequence of the double- stranded target site that is complementary to the sequence of the target domain, and thus with the strand complementary to the strand that comprises the PAM sequence.
  • the targeting domain of the gRNA typically does not include the PAM sequence.
  • the location of the PAM may be 5' or 3' of the target domain sequence, depending on the nuclease employed.
  • the PAM is typically 3' of the target domain sequences for Cas9 nucleases, and 5' of the target domain sequence for Cas12a nucleases.
  • the targeting domain may comprise a nucleotide sequence that corresponds to the sequence of the target domain, i.e., the DNA sequence directly adjacent to the PAM sequence (e.g., 5' of the PAM sequence for Cas9 nucleases, or 3' of the PAM sequence for Cas12a nucleases).
  • the targeting domain sequence typically comprises between 17 and 30 nucleotides and corresponds fully with the target domain sequence (i.e., without any mismatch nucleotides), or may comprise one or more, but typically not more than 4, mismatches.
  • the targeting domain is part of an RNA molecule, the gRNA, it will typically comprise ribonucleotides, while the DNA targeting domain will comprise deoxyribonucleotides.
  • FIG. 1 An exemplary illustration of a Cas9 target site, comprising a 22 nucleotide target domain, and an NGG PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target domain (and thus base-pairs with full complementarity with the DNA strand complementary to the strand comprising the target domain and PAM) is provided below:
  • FIG. 1 An exemplary illustration of a Cas12a target site, comprising a 22 nucleotide target domain, and a TTN PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target domain (and thus base-pairs with full complementarity with the DNA strand complementary to the strand comprising the target domain and PAM) is provided below:
  • the Cas12a PAM sequence is 5'-T-T-T-V-3'.
  • the length and complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA/Cas9 molecule complex with a target nucleic acid.
  • the targeting domain of a gRNA provided herein is 5 to 50 nucleotides in length. In some embodiments, the targeting domain is 15 to 25 nucleotides in length. In some embodiments, the targeting domain is 18 to 22 nucleotides in length. In some embodiments, the targeting domain is 19-21 nucleotides in length. In some embodiments, the targeting domain is 15 nucleotides in length.
  • the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length.
  • the targeting domain fully corresponds, without mismatch, to a target domain sequence provided herein, or a part thereof.
  • the targeting domain of a gRNA provided herein comprises 1 mismatch relative to a target domain sequence provided herein.
  • the targeting domain comprises 2 mismatches relative to the target domain sequence.
  • the target domain comprises 3 mismatches relative to the target domain sequence.
  • a targeting domain comprises a core domain and a secondary targeting domain, e.g., as described in PCT Publication No. W02015/157070, which is incorporated by reference in its entirety.
  • the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the most 3' 8 to 13 nucleotides of the targeting domain).
  • the secondary domain is positioned 5' to the core domain.
  • the core domain corresponds fully with the target domain sequence, or a part thereof.
  • the core domain may comprise one or more nucleotides that are mismatched with the corresponding nucleotide of the target domain sequence.
  • the gRNA comprises a first complementarity domain and a second complementarity domain, wherein the first complementarity domain is complementary with the second complementarity domain, and, at least in some embodiments, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions.
  • the first complementarity domain is 5 to 30 nucleotides in length.
  • the first complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain.
  • the 5' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.
  • the central subdomain is 1, 2, or 3, e.g., 1, nucleotide in length.
  • the 3' subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a 5. pyogenes, S. aureus or 5. thermophilus, first complementarity domain.
  • a linking domain may serve to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA.
  • the linking domain can link the first and second complementarity domains covalently or non-covalently.
  • the linkage is covalent.
  • the linking domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementarity domain.
  • the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the linking domain comprises at least one non-nucleotide bond, e.g., as disclosed in PCT Publication No. WO2018/126176, the entire contents of which are incorporated herein by reference.
  • the second complementarity domain is complementary, at least in part, with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions.
  • the second complementarity domain can include a sequence that lacks complementarity with the first complementarity domain, e.g., a sequence that loops out from the duplexed region.
  • the second complementarity domain is 5 to 27 nucleotides in length. In some embodiments, the second complementarity domain is longer than the first complementarity region.
  • the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the second complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain.
  • the 5' subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the central subdomain is 1, 2, 3, 4 or 5, e.g., 3, nucleotides in length.
  • the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.
  • the 5' subdomain and the 3' subdomain of the first complementarity domain are respectively, complementary, e.g., fully complementary, with the 3' subdomain and the 5' subdomain of the second complementarity domain.
  • the proximal domain is 5 to 20 nucleotides in length. In some embodiments, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In an embodiment, it has at least 50% homology with a proximal domain from S. pyogenes, S. aureus, or S. thermophilus.
  • tail domains are suitable for use in gRNAs.
  • the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.
  • the tail domain nucleotides are from or share homology with a sequence from the 5' end of a naturally occurring tail domain.
  • the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.
  • the tail domain is absent or is 1 to 50 nucleotides in length.
  • the tail domain can share homology with or be derived from a naturally occurring proximal tail domain.
  • the tail domain has at least 50% homology/identity with a tail domain from S. pyogenes, S. aureus or S. thermophilus.
  • the tail domain includes nucleotides at the 3' end that are related to the method of in vitro or in vivo transcription.
  • a gRNA provided herein comprises: a first strand comprising, e.g., from 5' to 3': a targeting domain (which corresponds to a target domain in the CD38 gene); and a first complementarity domain; and a second strand, comprising, e.g., from 5' to 3': optionally, a 5' extension domain; a second complementarity domain; a proximal domain; and optionally, a tail domain.
  • any of the gRNAs provided herein comprise one or more nucleotides that are chemically modified. Chemical modifications of gRNAs have previously been described, and suitable chemical modifications include any modifications that are beneficial for gRNA function and do not measurably increase any undesired characteristics, e.g., off-target effects, of a given gRNA.
  • Suitable chemical modifications include, for example, those that make a gRNA less susceptible to endo- or exonuclease catalytic activity, and include, without limitation, phosphorothioate backbone modifications, 2'-O-Me-modifications (e.g., at one or both of the 3' and 5' termini), 2'F-modifications, replacement of the ribose sugar with the bicyclic nucleotide-cEt, 3 'thioPACE (MSP) modifications, or any combination thereof.
  • Additional suitable gRNA modifications will be apparent to the skilled artisan based on this disclosure, and such suitable gRNA modifications include, without limitation, those described, e.g., in Rahdar et al. PNAS (2015) 112 (51) E7110-E7117 and Hendel et al., Nat Biotechnol. (2015); 33(9): 985-989, each of which is incorporated herein by reference in its entirety.
  • a gRNA provided herein may comprise one or more 2'-O modified nucleotide, e.g., a 2'-O-methyl nucleotide.
  • the gRNA comprises a 2'-O modified nucleotide, e.g., 2'-O-methyl nucleotide at the 5' end of the gRNA.
  • the gRNA comprises a 2'-O modified nucleotide, e.g., 2'-O- methyl nucleotide at the 3' end of the gRNA.
  • the gRNA comprises a 2'-O-modified nucleotide, e.g., a 2'-O-methyl nucleotide at both the 5' and 3' ends of the gRNA.
  • the gRNA is 2'-O-modified, e.g. 2'-O-methyl-modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA.
  • the gRNA is 2'-O-modified, e.g.
  • the gRNA is 2'-O-modified, e.g.
  • the gRNA is 2'-O-modified, e.g.
  • the nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O-modified, e.g.
  • the 2'-O-methyl nucleotide comprises a phosphate linkage to an adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide comprises a phosphorothioate linkage to an adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide comprises a thioPACE linkage to an adjacent nucleotide.
  • a gRNA provided herein may comprise one or more 2'- O-modified and 3'phosphorous-modified nucleotide, e.g., a 2'-O-methyl 3 'phosphorothioate nucleotide.
  • the gRNA comprises a 2'-O-modified and
  • the gRNA comprises a 2'-O-modified and 3'phosphorous- modified, e.g., 2'-O-methyl 3 'phosphorothioate nucleotide at the 3' end of the gRNA.
  • the gRNA comprises a 2'-O-modified and 3'phosphorous-modified, e.g., 2'-O-methyl 3 'phosphorothioate nucleotide at the 5' and 3' ends of the gRNA.
  • the gRNA comprises a backbone in which one or more non-bridging oxygen atoms has been replaced with a sulfur atom.
  • the gRNA is 2'-O- modified and 3'phosphorous-modified, e.g. 2'-O-methyl 3'phosphorothioate-modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA.
  • the gRNA is 2'-O-modified and 3'phosphorous-modified, e.g.
  • the gRNA is 2'-O-modified and 3'phosphorous-modified, e.g.
  • the gRNA is 2'-O-modified and 3'phosphorous-modified, e.g.
  • the nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O-modified and 3'phosphorous-modified, e.g.
  • a gRNA provided herein may comprise one or more 2'- O-modified and 3'-phosphorous-modified, e.g., 2'-O-methyl 3 'thioPACE nucleotide.
  • the gRNA comprises a 2'-O-modified and 3'phosphorous-modified, e.g., 2'-O-methyl 3'thioPACE nucleotide at the 5' end of the gRNA.
  • the gRNA comprises a 2'-O-modified and 3'phosphorous-modified, e.g., 2'-O-methyl 3'thioPACE nucleotide at the 3' end of the gRNA.
  • the gRNA comprises a 2'-O-modified and 3'phosphorous-modified, e.g., 2'-O-methyl 3'thioPACE nucleotide at the 5' and 3' ends of the gRNA.
  • the gRNA comprises a backbone in which one or more non-bridging oxygen atoms have been replaced with a sulfur atom and one or more non-bridging oxygen atoms have been replaced with an acetate group.
  • the gRNA is 2'-O-modified and 3'phosphorous-modified, e.g.
  • the gRNA is 2'-O-modified and 3'phosphorous-modified, e.g. 2'-O-methyl 3 'thioPACE-modified at the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA.
  • the gRNA is 2'-O-modified and 3'phosphorous-modified, e.g. 2'-O-methyl 3 'thioPACE-modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA.
  • the gRNA is 2'-O-modified and 3'phosphorous-modified, e.g.
  • the nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O-modified and 3'phosphorous-modified, e.g.
  • a gRNA provided herein comprises a chemically modified backbone.
  • the gRNA comprises a phosphorothioate linkage.
  • one or more non-bridging oxygen atoms have been replaced with a sulfur atom.
  • the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each comprise a phosphorothioate linkage.
  • the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage.
  • the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage.
  • the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and at the fourth nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage.
  • the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage.
  • a gRNA provided herein comprises a thioPACE linkage.
  • the gRNA comprises a backbone in which one or more non- bridging oxygen atoms have been replaced with a sulfur atom and one or more non-bridging oxygen atoms have been replaced with an acetate group.
  • the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each comprise a thioPACE linkage.
  • the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a thioPACE linkage.
  • the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a thioPACE linkage.
  • the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and at the fourth nucleotide from the 3' end of the gRNA each comprise a thioPACE linkage.
  • the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a thioPACE linkage.
  • a gRNA described herein comprises one or more 2'-O- methyl- 3 '-phosphoro thio ate nucleotides, e.g., at least 1, 2, 3, 4, 5, or 6 2'-O-methyl-3'- phosphorothioate nucleotides.
  • a gRNA described herein comprises modified nucleotides (e.g., 2'-O-methyl-3'-phosphorothioate nucleotides) at one or more of the three terminal positions and the 5' end and/or at one or more of the three terminal positions and the 3' end.
  • the gRNA may comprise one or more modified nucleotides, e.g., as described in PCT Publication Nos. WO2017/214460, WO2016/089433, and WO2016/164356, which are incorporated by reference their entirety.
  • the CD38-targeting gRNAs provided herein can be delivered to a cell in any manner suitable.
  • CRISPR/Cas systems comprising an RNP including a gRNA bound to an RNA-guided nuclease
  • exemplary suitable methods include, without limitation, electroporation of RNP into a cell, electroporation of mRNA encoding a Cas nuclease and a gRNA into a cell, various protein or nucleic acid transfection methods, and delivery of encoding RNA or DNA via viral vectors, such as, for example, retroviral (e.g., lentiviral) vectors.
  • retroviral e.g., lentiviral
  • the present disclosure provides a number of CD38 target sites and corresponding gRNAs that are useful for targeting an RNA-guided nuclease to human CD38.
  • Table 1 below illustrates preferred target domains in the human endogenous CD38 gene that can be bound by gRNAs described herein.
  • the exemplary target sequences of human CD38 shown in Table 1, in some embodiments, are for use with a Cas9 nuclease, e.g., SpCas9.
  • Exemplary Cas9 target site sequences of human CD38 are provided, as are exemplary gRNA targeting domain sequences useful for targeting such sites.
  • the first sequence represents the DNA target domain sequence
  • the second sequence represents the complement thereof
  • the third sequence represents the reverse complement thereof
  • the fourth sequence represents an exemplary targeting domain sequence of a gRNA that can be used to target the respective target site.
  • Exemplary Cas9 target site sequences of human CD38 are provided, as are exemplary gRNA targeting domain sequences useful for targeting such sites.
  • the first sequence represents the DNA target domain sequence
  • the second sequence represents the complement thereof
  • the third sequence represents the reverse complement thereof
  • the fourth sequence represents an exemplary targeting domain sequence of a gRNA that can be used to target the respective target site.
  • the present disclosure provides exemplary CD38 targeting gRNAs that are useful for targeting an RNA-guided nuclease to human CD38.
  • Table 3 below illustrates preferred targeting domains for use in gRNAs targeting Cas9 nucleases to human endogenous CD38 gene.
  • the exemplary target sequences of human CD38 shown in Table 3, in some embodiments, are for use with a Cas9 nuclease, e.g., SpCas9.
  • Table 3 Exemplary targeting domain sequences of gRNAs that target human CD38 are provided.
  • the present disclosure provides a number of CD38 target sites and corresponding gRNAs that are useful for targeting an RNA-guided nuclease to human CD38.
  • Table 4 below illustrates preferred target domains in the human endogenous CD38 gene that can be bound by gRNAs described herein.
  • the exemplary target sequences of human CD38 shown in Table 4, in some embodiments, are for use with a Cpf1 nuclease.
  • Exemplary Cas12a/Cpf1 target site sequences of human CD38 are provided, as are exemplary gRNA targeting domain sequences useful for targeting such sites.
  • the first sequence represents the DNA target domain sequence
  • the second sequence represents the complement thereof
  • the third sequence represents the reverse complement thereof
  • the fourth sequence represents an exemplary targeting domain sequence of a gRNA that can be used to target the respective target site.
  • the present disclosure provides exemplary CD38 targeting gRNAs that are useful for targeting an RNA-guided nuclease to human CD38.
  • Table 5 illustrates preferred targeting domains for use in gRNAs targeting Cas9 nucleases to human endogenous CD38 gene.
  • the exemplary target sequences of human CD38 shown in Table 5, in some embodiments, are for use with a Cpf 1 nuclease.
  • Some aspects of this disclosure provide genetically engineered cells comprising a modification in their genome that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • the modification in the genome of the cell is a mutation in a genomic sequence encoding CD38.
  • the modification is affected via genome editing, e.g., using a Cas nuclease and a gRNA targeting a CD38 target site provided herein or comprising a targeting domain sequence provided herein.
  • compositions, methods, strategies, and treatment modalities provided herein may be applied to any cell or cell type, some exemplary cells and cell types that are particularly suitable for genomic modification in the CD38 gene according to aspects of this invention are described in more detail herein. The skilled artisan will understand, however, that the provision of such examples is for the purpose of illustrating some specific embodiments, and additional suitable cells and cell types will be apparent to the skilled artisan based on the present disclosure, which is not limited in this respect.
  • Some aspects of this disclosure provide genetically engineered hematopoietic cells comprising a modification in their genome that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • the genetically engineered cells comprising a modification in their genome results in reduced cell surface expression of CD38 and/or reduced binding by an immunotherapeutic agent targeting CD38, e.g., as compared to a hematopoietic cell of the same cell type but not comprising a genomic modification.
  • a hematopoietic cell is a hematopoietic stem cell (HSC).
  • the hematopoietic cell is a hematopoietic progenitor cell (HPC). In some embodiments, the hematopoietic cell is a hematopoietic stem or progenitor cell.
  • HPC hematopoietic progenitor cell
  • the cells are CD34+.
  • the cell is a hematopoietic cell.
  • the cell is a hematopoietic stem cell.
  • the cell is a hematopoietic progenitor cell.
  • the cell is an immune effector cell.
  • the cell is a lymphocyte.
  • the cell is a T-lymphocyte.
  • the cell is a NK cell.
  • the cell is a stem cell.
  • the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a mesenchymal stem cell, or a tissue-specific stem cell.
  • ESC embryonic stem cell
  • iPSC induced pluripotent stem cell
  • mesenchymal stem cell or a tissue-specific stem cell.
  • the cells are comprised in a population of cells which is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient and to generate differentiated progeny of all blood lineage cell types in the recipient.
  • the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 50%.
  • the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 60%.
  • the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 70%. In some embodiments, the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 80%. In some embodiments, the cell population is characterized by the ability to engraft CD38-edited hematopoietic stem cells in the bone marrow of a recipient at an efficiency of at least 90%. In some embodiments, the cell population comprises CD38- edited hematopoietic stem cells that are characterized by a differentiation potential that is equivalent to the differentiation potential of non-edited hematopoietic stem cells.
  • a hematopoietic cell e.g., an HSC or HPC
  • a hematopoietic cell comprising a modification in their genome that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38
  • a nuclease and/or a gRNA targeting human CD38 as described herein. It will be understood that such a cell can be created by contacting the cell with the nuclease and/or the gRNA, or the cell can be the daughter cell of a cell that was contacted with the nuclease and/or gRNA.
  • a cell described herein e.g., a genetically engineered HSC or HPC
  • a cell described herein is capable of populating the HSC or HPC niche and/or of reconstituting the hematopoietic system of a subject.
  • a cell described herein e.g., an HSC or HPC
  • a genetically engineered hematopoietic cell provided herein, or its progeny can differentiate into all blood cell lineages, preferably without any differentiation bias as compared to a hematopoietic cell of the same cell type, but not comprising a genomic modification that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • chimerism The level of engrafted donor cells or descendants thereof relative to host cells in a given tissue or niche is referred to herein as chimerism.
  • a cell described herein e.g., an HSC or HPC
  • a cell described herein is capable of engrafting in a human subject and does not exhibit any difference in chimerism as compared to a hematopoietic cell of the same cell type, but not comprising a genomic modification that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • a cell described herein e.g., an HSC or HPC
  • a cell described herein is capable of engrafting in a human subject exhibits no more than a 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% difference in chimerism as compared to a hematopoietic cell of the same cell type, but not comprising a genomic modification that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • a genetically engineered cell provided herein comprises only one genomic modification, e.g., a genomic modification that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38. It will be understood that the gene editing methods provided herein may result in genomic modifications in one or both alleles of a target gene. In some embodiments, genetically engineered cells comprising a genomic modification in both alleles of a given genetic locus are preferred.
  • a genetically engineered cell comprises two or more genomic modifications, e.g., one or more genomic modifications in addition to a genomic modification that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38.
  • a genetically engineered cell comprises a genomic modification that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38, and further comprises an expression construct that encodes a chimeric antigen receptor, e.g., in the form of an expression construct encoding the CAR integrated in the genome of the cell.
  • the CAR comprises a binding domain, e.g., an antibody fragment, that binds CD38.
  • the immune effector cell is a lymphocyte.
  • the immune effector cell is a T-lymphocyte.
  • the T-lymphocyte is an alpha/beta T-lymphocyte.
  • the T-lymphocyte is a gamma/delta T-lymphocyte.
  • the immune effector cell is a natural killer T (NKT cell).
  • the immune effector cell is a natural killer (NK) cell.
  • the immune effector cell does not express an endogenous transgene, e.g., a transgenic protein. In some embodiments, the immune effector cell expresses a chimeric antigen receptor (CAR). In some embodiments, the immune effector cell expresses a CAR targeting CD38. In some embodiments, the immune, effector cell does not express a CAR targeting CD38.
  • CAR chimeric antigen receptor
  • a genetically engineered cell comprises a genomic modification that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38, and does not comprise an expression construct that encodes an exogenous protein, e.g., does not comprise an expression construct encoding a CAR.
  • a genetically engineered cell provided herein expresses substantially no CD38 protein, e.g., expresses no CD38 protein that can be measured by a suitable method, such as an immuno staining method.
  • a genetically engineered cell provided herein expresses substantially no wild-type CD38 protein, but expresses a mutant CD38 protein variant, e.g., a variant not recognized by an immunotherapeutic agent targeting CD38, e.g., a CAR-T cell therapeutic, or an anti-CD38 antibody, antibody fragment, or antibody-drug conjugate (ADC).
  • an immunotherapeutic agent targeting CD38 e.g., a CAR-T cell therapeutic, or an anti-CD38 antibody, antibody fragment, or antibody-drug conjugate (ADC).
  • the genetically engineered cells provided herein are hematopoietic cells, e.g., hematopoietic stem cells, hematopoietic progenitor cell (HPC), hematopoietic stem or progenitor cell.
  • hematopoietic stem cells e.g., hematopoietic stem cells, hematopoietic progenitor cell (HPC), hematopoietic stem or progenitor cell.
  • HPC hematopoietic progenitor cell
  • Hematopoietic stem cells are cells characterized by pluripotency, self-renewal properties, and/or the ability to generate and/or reconstitute all lineages of the hematopoietic system, including both myeloid and lymphoid progenitor cells that further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively.
  • myeloid cells e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc
  • lymphoid cells e.g., T cells, B cells, NK cells
  • HSCs are characterized by the expression of one or more cell surface markers, e.g., CD34 (e.g., CD34+), which can be used for the identification and/or isolation of HSCs, and absence of cell surface markers associated with commitment to a cell lineage.
  • a genetically engineered cell e.g., genetically engineered HSC described herein does not express one or more cell-surface markers typically associated with HSC identification or isolation, expresses a reduced amount of the cell-surface markers, or expresses a variant cell-surface marker not recognized by an immunotherapeutic agent targeting the cell- surface marker, but nevertheless is capable of self-renewal and can generate and/or reconstitute all lineages of the hematopoietic system.
  • a population of genetically engineered cells described herein comprises a plurality of genetically engineered hematopoietic stem cells. In some embodiments, a population of genetically engineered cells described herein comprises a plurality of genetically engineered hematopoietic progenitor cells. In some embodiments, a population of genetically engineered cells described herein comprises a plurality of genetically engineered hematopoietic stem cells and a plurality of genetically engineered hematopoietic progenitor cells.
  • the genetically engineered HSCs are obtained from a subject, such as a human subject. Methods of obtaining HSCs are described, e.g., in PCT Application No. US2016/057339, which is herein incorporated by reference in its entirety.
  • the HSCs are peripheral blood HSCs.
  • the mammalian subject is a non-human primate, a rodent (e.g., mouse or rat), a bovine, a porcine, an equine, or a domestic animal.
  • the HSCs are obtained from a human subject, such as a human subject having a hematopoietic malignancy.
  • the HSCs are obtained from a healthy donor. In some embodiments, the HSCs are obtained from the subject to whom the immune cells expressing the chimeric receptors will be subsequently administered. HSCs that are administered to the same subject from which the cells were obtained are referred to as autologous cells, whereas HSCs that are obtained from a subject who is not the subject to whom the cells will be administered are referred to as allogeneic cells.
  • a population of genetically engineered cells is a heterogeneous population of cells, e.g. heterogeneous population of genetically engineered cells containing different CD38 mutations.
  • at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of copies of a gene encoding CD38 in the population of genetically engineered cells comprise a mutation effected by a genome editing approach described herein, e.g., by a CRISPR/Cas system using a gRNA provided herein.
  • a population of genetically engineered cells can comprise a plurality of different CD38 mutations and each mutation of the plurality may contribute to the percent of copies of CD38 in the population of cells that have a mutation.
  • the expression of CD38 on the genetically engineered hematopoietic cell is compared to the expression of CD38 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • the genetic engineering results in a reduction in the expression level of CD38 by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% as compared to the expression of CD38 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • the genetically engineered hematopoietic cell expresses less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD38 as compared to a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • a naturally occurring hematopoietic cell e.g., a wild-type counterpart
  • the genetic engineering as described herein results in a reduction in the expression level of wild-type CD38 by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% as compared to the expression of the level of wild-type CD38 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • a naturally occurring hematopoietic cell e.g., a wild-type counterpart
  • the genetically engineered hematopoietic cell expresses less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD38 as compared to a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • a naturally occurring hematopoietic cell e.g., a wild-type counterpart
  • the genetic engineering as described herein results in a reduction in the expression level of wild-type lineage-specific cell surface antigen (e.g., CD38) by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% as compared to a suitable control (e.g., a cell or plurality of cells).
  • a suitable control e.g., a cell or plurality of cells.
  • the suitable control comprises the level of the wild-type lineage-specific cell surface antigen measured or expected in a plurality of non-engineered cells from the same subject. In some embodiments, the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a plurality of cells from a healthy subject. In some embodiments, the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a population of cells from a pool of healthy individuals (e.g., 10, 20, 50, or 100 individuals).
  • the suitable control comprises the level of the wild-type lineage-specific cell surface antigen measured or expected in a subject in need of a treatment described herein, e.g., an anti-CD38 therapy, e.g., wherein the subject has a cancer, wherein cells of the cancer express CD38.
  • a method of genetically engineering cells described herein comprises a step of providing a wild-type cell, e.g., a wild-type hematopoietic stem or progenitor cell.
  • the wile-type cell is an un-edited cell comprising (e.g., expressing) two functional copies of a gene encoding CD38.
  • the cell comprises a CD38 gene sequence according to SEQ ID NO: 157.
  • the cell comprises a CD38 gene sequence encoding a CD38 protein that is encoded in SEQ ID NO: 156, e.g., the CD38 gene sequence may comprise one or more silent mutations relative to SEQ ID NO: 157.
  • the cell used in the method is a naturally occurring cell or a non-engineered cell.
  • the wild-type cell expresses CD38, or gives rise to a more differentiated cell that expresses CD38 at a level comparable to (or within 90%-110%, 80%-120%, 70%-130%, 60-140%, or 50%-150% of) a cell line expressing CD38, such as Daudi, HDLM-2, MOLT-4,, REH, Karpas-707, RPMI-8226, U- 266/70, U-698, A549 cells.
  • the wild-type cell binds an antibody that binds CD38 (e.g., an anti-CD38 antibody, e.g., daratumumab, isatuximab), or gives rise to a more differentiated cell that binds such an antibody at a level comparable to (or within 90% - 110%, 80%-120%, 70%-130%, 60-140%, or 50%-150% of) binding of the antibody to a cell line expressing CD38, Daudi, HDLM-2, MOLT-4,, REH, Karpas-707, RPMI-8226, U- 266/70, U-698, A549 cells.
  • Antibody binding may be measured, for example, by flow cytometry or immunohistochemistry.
  • a gRNA provided herein can be used in combination with a second gRNA, e.g., for targeting a CRISPR/Cas nuclease to two sites in a genome.
  • a second gRNA e.g., for targeting a CRISPR/Cas nuclease to two sites in a genome.
  • the disclosure provides various combinations of gRNAs and related CRISPR systems, as well as cells created by genome editing methods using such combinations of gRNAs and related CRISPR systems.
  • the CD38 gRNA binds a different nuclease than the second gRNA.
  • the CD38 gRNA may bind Cas9 and the second gRNA may bind Cas12a, or vice versa.
  • the first gRNA is a CD38 gRNA provided herein (e.g., a gRNA provided in any of Tables 1-5 or a variant thereof) and the second gRNA targets a lineage- specific cell-surface antigen chosen from: BCMA, CD19, CD20, CD30, ROR1, B7H6, B7H3, CD23, CD33, CD38, C-type lectin like molecule-1, CS1, IL-5, L1-CAM, PSCA, PSMA, CD138, CD133, CD70, CD5, CD6, CD7, CD13, NKG2D, NKG2D ligand, CLEC12A, CD11, CD123, CD386, CD30, CD34, CD14, CD66b, CD41, CD61, CD62, CD235a, CD146, CD326, LMP2, CD22, CD382, CD10, CD3/TCR, CD79/BCR, and CD26.
  • a lineage- specific cell-surface antigen chosen from: BCMA, CD19, CD20, CD
  • the first gRNA is a CD38 gRNA provided herein (e.g., a gRNA provided in any one of Tables 1-5 or a variant thereof) and the second gRNA targets a lineage- specific cell-surface antigen associated with a neoplastic or malignant disease or disorder, e.g., with a specific type of cancer, such as, without limitation, CD20, CD22 (Non- Hodgkin's lymphoma, B-cell lymphoma, chronic lymphocytic leukemia (CLL)), CD382 (B- cell CLL), CD33 (Acute myelogenous leukemia (AML)), CD 10 (gp100) (Common (pre-B) acute lymphocytic leukemia and malignant melanoma), CD3/T-cell receptor (TCR) (T-cell lymphoma and leukemia), CD79/B-cell receptor (BCR) (B-cell lymphoma and leukemia),
  • a specific type of cancer
  • the first gRNA is a CD38 gRNA provided herein (e.g., a gRNA provided in any one of Tables 1-5 or a variant thereof) and the second gRNA targets a lineage- specific cell-surface antigen chosen from: CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDwl2, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b
  • the second gRNA is a gRNA disclosed in any of W02017/066760, WO2019/046285, WO/2018/160768, or Borot et al. PNAS (2019) 116 (24) 11978-11987, each of which is incorporated herein by reference in its entirety.
  • Some aspects of this disclosure provide methods comprising administering an effective number of genetically engineered cells as described herein, comprising a modification in their genome that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38, to a subject in need thereof.
  • a subject in need thereof is, in some embodiments, a subject undergoing or about to undergo an immunotherapy targeting CD38.
  • a subject in need thereof is, in some embodiments, a subject having or having been diagnosed with, a malignancy characterized by expression of CD38 on malignant cells.
  • a subject having such a malignancy may be a candidate for immunotherapy targeting CD38, but the risk of detrimental on-target, off-disease effects may outweigh the benefit, expected or observed, to the subject.
  • administration of genetically engineered cells as described herein results in an amelioration of the detrimental on-target, off-disease effects, as the genetically engineered cells provided herein are not targeted efficiently by an immunotherapeutic agent targeting CD38.
  • the malignancy is a hematologic malignancy, or a cancer of the blood.
  • the malignancy is a lymphoid malignancy.
  • lymphoid malignancies are associated with the inappropriate production, development, and/or function of lymphoid cells, such as lymphocytes of the T lineage or the B lineage.
  • the malignancy is characterized or associated with cells that express CD38 on the cell surface.
  • the malignancy is associated with aberrant T lymphocytes, such as a T-lineage cancer, e.g., a T cell leukemia or a T-cell lymphoma.
  • T cell leukemias and T-cell lymphomas include, without limitation, T-lineage Acute Lymphoblastic Leukemia (T-ALL), Hodgkin's lymphoma, or a non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), large granular lymphocytic leukemia, adult T-cell leukemia/lymphoma (ATLL), T-cell prolymphocytic leukemia (T-PLL), T-cell chronic lymphocytic leukemia, , T- prolymphocytic leukemia, T-cell lymphocytic leukemia, B-cell chronic lymphocytic leukemia, mantle cell lymph
  • the malignancy is associated with aberrant B lymphocytes, such as a B-lineage cancer, e.g., a B-cell leukemia or a B-cell lymphoma.
  • a B-lineage cancer e.g., a B-cell leukemia or a B-cell lymphoma.
  • the malignancy is B-lineage Acute Lymphoblastic Leukemia (B-ALL) or chronic lymphocytic leukemia (B-CLL).
  • the hematopoietic malignancy associated with or characterized by expression of CD38 is multiple myeloma, B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia, chronic myeloid leukemia, Waldenstrom macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, spherical leukemia, chronic myelogenous leukemia, follicular lymphoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), NK cell leukemia, and plasma cell leukemia.
  • B-cell chronic lymphocytic leukemia B-cell acute lymphoblastic leukemia
  • chronic myeloid leukemia Waldenstrom macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, spherical leukemia, chronic myelogenous leukemia, follicular lymphoma,
  • malignancies that are considered to be relapsed and/or refractory, such as relapsed or refractory hematological malignancies.
  • a subject in need thereof is, in some embodiments, a subject undergoing or that will undergo an immune effector cell therapy targeting CD38, e.g., CAR-T cell therapy, wherein the immune effector cells express a CAR targeting CD38, and wherein at least a subset of the immune effector cells also express CD38 on their cell surface.
  • an immune effector cell therapy targeting CD38 e.g., CAR-T cell therapy
  • the immune effector cells express a CAR targeting CD38
  • at least a subset of the immune effector cells also express CD38 on their cell surface.
  • the term "fratricide” refers to self-killing. For example, cells of a population of cells kill or induce killing of cells of the same population.
  • cells of the immune effector cell therapy kill or induce killing of other cells of the immune effector cell therapy.
  • fratricide ablates a portion of or the entire population of immune effector cells before a desired clinical outcome, e.g., ablation of malignant cells expressing CD38 within the subject, can be achieved.
  • using genetically engineered immune effector cells, as provided herein, e.g., immune effector cells that do not express CD38 or do not express a CD38 variant recognized by the CAR, as the immune effector cells forming the basis of the immune effector cell therapy will avoid such fratricide and the associated negative impact on therapy outcome.
  • genetically engineered immune effector cells may be further modified to also express the CD38-targeting CAR.
  • the immune effector cells may be lymphocytes, e.g., T-lymphocytes, such as, for example alpha/beta T- lymphocytes, gamma/delta T-lymphocytes, or natural killer T cells.
  • the immune effector cells may be natural killer (NK) cells.
  • an effective number of genetically engineered cells as described herein, comprising a modification in their genome that results in a loss of expression of CD38, or expression of a variant form of CD38 that is not recognized by an immunotherapeutic agent targeting CD38 is administered to a subject in need thereof, e.g., to a subject undergoing or that will undergo an immunotherapy targeting CD38, wherein the immunotherapy is associated or is at risk of being associated with a detrimental on-target, off-disease effect, e.g., in the form of cytotoxicity towards healthy cells in the subject that express CD38.
  • an effective number of such genetically engineered cells may be administered to the subject in combination with the anti-CD38 immunotherapeutic agent.
  • agents e.g., CD38-modified cells and an anti-CD38 immunotherapeutic agent
  • the cells and the agent may be administered at the same time or at different times, e.g., in temporal proximity.
  • the cells and the agent may be admixed or in separate volumes or dosage forms.
  • administration in combination includes administration in the same course of treatment, e.g., in the course of treating a subject with an anti-CD38 immunotherapy, the subject may be administered an effective number of genetically engineered, CD38-modified cells concurrently or sequentially, e.g., before, during, or after the treatment, with the anti-CD38 immunotherapy.
  • the immunotherapeutic agent that targets CD38 as described herein is an immune cell that expresses a chimeric antigen receptor, which comprises an antigen-binding fragment (e.g., a single-chain antibody) capable of binding to CD38.
  • the immune cell may be, e.g., a T cell (e.g., a CD4+ or CD8+ T cell) or an NK cell.
  • a Chimeric Antigen Receptor can comprise a recombinant polypeptide comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain, e.g., one derived from a stimulatory molecule.
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, such as 4-1BB (i.e., CD137), CD27, and/or CD28, or fragments of those molecules.
  • the extracellular antigen binding domain of the CAR may comprise a CD38-binding antibody fragment.
  • the antibody fragment can comprise one or more CDRs, the variable regions (or portions thereof), the constant regions (or portions thereof), or combinations of any of the foregoing.
  • Amino acid and nucleic acid sequences of an exemplary heavy chain variable region and light chain variable region of an anti-human CD38 antibody are provided, for example in Guo et al. Cell. & Mol. Immunol. (2020) 17: 430-432.
  • a chimeric antigen receptor typically comprises an antigen-binding domain, e.g., comprising an antibody fragment, fused to a CAR framework, which may comprise a hinge region (e.g., from CD8 or CD28), a transmembrane domain (e.g., from CD8 or CD28), one or more costimulatory domains (e.g., CD28 or 4- IBB), and a signaling domain (e.g., CD3zeta).
  • a hinge region e.g., from CD8 or CD28
  • a transmembrane domain e.g., from CD8 or CD28
  • costimulatory domains e.g., CD28 or 4- IBB
  • signaling domain e.g., CD3zeta
  • the number of genetically engineered cells provided herein e.g., HSCs, HPCs, or immune effector cells that are administered to a subject in need thereof, is within the range of 10 6 -10 11 .
  • amounts below or above this exemplary range are also within the scope of the present disclosure.
  • the number of genetically engineered cells provided herein, e.g., HSCs, HPCs, or immune effector cells that are administered to a subject in need thereof is about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , or about 10 11 .
  • the number of genetically engineered cells provided herein, e.g., HSCs, HPCs, or immune effector cells that are administered to a subject in need thereof is within the range of 10 6 -10 9 , within the range of 10 6 -10 8 , within the range of 10 7 -10 9 , within the range of about 10 7 -10 10 , within the range of 10 8 -10 10 , or within the range of 10 9 - 10 11 .
  • the immunotherapeutic agent that targets CD38 is an antibody-drug conjugate (ADC).
  • ADC may be a molecule comprising an antibody or antigen-binding fragment thereof conjugated to a toxin or drug molecule. Binding of the antibody or fragment thereof to the corresponding antigen allows for delivery of the toxin or drug molecule to a cell that presents the antigen on the its cell surface (e.g., target cell), thereby resulting in death of the target cell.
  • Suitable antibodies and antibody fragments binding CD38 will be apparent to those of ordinary skill in the art, and include, for example, those described in PCT Publication Nos.
  • Toxins or drugs compatible for use in antibody-drug conjugates are known in the art and will be evident to one of ordinary skill in the art. See, e.g., Peters et al. Biosci. Rep. (2015) 35(4): e00225; Beck et al. Nature Reviews Drug Discovery (2017) 16:315-337; Marin-Acevedo et al. J. Hematol. Oncol. (2016)11: 8; Elgundi et al. Advanced Drug Delivery Reviews (2017) 122: 2-19.
  • the antibody-drug conjugate may further comprise a linker (e.g., a peptide linker, such as a cleavable linker) attaching the antibody and drug molecule.
  • a linker e.g., a peptide linker, such as a cleavable linker
  • Suitable toxins or drugs for antibody-drug conjugates include, without limitation, the toxins and drugs comprised in brentuximab vedotin, glembatumumab vedotin/CDX-011, depatuxizumab mafodotin/ ABT-414, PSMA ADC, polatuzumab vedotin/RG7596/DCDS4501A, denintuzumab mafodotin/SGN-CD19A, AGS-16C3F, CDX- 014, RG7841/DLYE5953A, RG7882/DMUC406A, RG7986/DCDS0780A, SGN-LIV1A, enfortumab vedotin/ASG-22ME, AG-15ME, AGS67E, telisotuzumab vedotin/ ABBV-399, ABBV-221, ABBV-085, GSK-2857916
  • binding of the antibody-drug conjugate to the epitope of the cell-surface lineage- specific protein induces internalization of the antibody-drug conjugate, and the drug (or toxin) may be released intracellularly.
  • binding of the antibody-drug conjugate to the epitope of a cell-surface lineage- specific protein induces internalization of the toxin or drug, which allows the toxin or drug to kill the cells expressing the lineage- specific protein (target cells).
  • binding of the antibody-drug conjugate to the epitope of a cell-surface lineage- specific protein induces internalization of the toxin or drug, which may regulate the activity of the cell expressing the lineage- specific protein (target cells).
  • the type of toxin or drug used in the antibody-drug conjugates described herein is not limited to any specific type.
  • the target domains and gRNAs indicated in Tables 1-5 were designed by manual inspection for a PAM sequence for an applicable nuclease, e.g., Cas9, Cpf1, with close proximity to the target region and prioritized according to predicted specificity by minimizing potential off-target sites in the human genome with an online search algorithm (e.g., the Benchling algorithm, Doench et al 2016, Hsu et al 2013). All designed synthetic sgRNAs were produced with chemically modified nucleotides at the three terminal positions at both the 5' and 3' ends. Modified nucleotides contained 2'-O-methyl-3'-phosphorothioate (abbreviated as "ms") and the ms-sgRNAs were HPLC-purified. Cas9 protein was purchased from Synthego.
  • an applicable nuclease e.g., Cas9, Cpf1
  • All designed synthetic sgRNAs were produced with chemically modified nucleotides at the three terminal positions at
  • CD34+ HSCs derived from mobilized peripheral blood (mPB) were purchased either from Hemacare or Fred Hutchinson Cancer Center and thawed according to manufacturer's instructions.
  • mPB mobilized peripheral blood
  • StemSpan CC110 cocktail StemSpan CC110 cocktail
  • CD34+ HSCs were electroporated using the Lonza Nucleofector 2 and the Human P3 Cell Nucleofection Kit (VPA-1002, Lonza).
  • Human CD34+ cells were electroporated with Cas9 protein and indicated CD38-targeting gRNAs, as described above.
  • the percentage editing was determined by % INDEL as assessed by TIDE and is short in Table 7 for example CD38 gRNAs. Editing efficiency was determined from the flow cytometric analysis.
  • the CD38 gRNA-edited cells may also be evaluated for surface expression of CD38 protein, for example by flow cytometry analysis (FACS).
  • FACS flow cytometry analysis
  • Live CD34+ HSCs are stained for CD38 using an anti-CD38 antibody and analyzed by flow cytometry on the Attune NxT flow cytometer (Life Technologies).
  • Cells in which the CD38 gene have been genetically modified show a reduction in CD38 expression as detected by FACS.
  • CD38 gRNAs were designed as described in Example 1 and shown in Tables 1-5.
  • T-lymphocytes such as Molt-4 cells
  • the cells were electroporated with pre-formed gRNA-nuclease (e.g., Cas9, Cpf1) RNP complex.
  • pre-formed gRNA-nuclease e.g., Cas9, Cpf1
  • 2e5 Molt-4 cells were electroporated with 3 ⁇ g Cas9:3 ⁇ g gRN A preformed RNP complex using a Lonza 4D-Nucleofector and P3 Primary Cell Kit.
  • the editing frequency was determined based on the percentage of alleles with indels compared to the wild-type sequence as assessed by Sanger sequence, followed by Tracking of Indels by Decomposition (TIDE) analysis (see, Brinkman et al. 2014; Hsiau et al. 2018).
  • the percentage editing was determined by % INDEL as assessed by TIDE and is shown in FIGURES 3A-3F and 5A-5G, and in Table 8 for examplary CD38 gRNAs. Table 8. Gene editing efficiency of CD38 gRNAs.
  • FIGURES 4A and 4B show flow cytometry analysis of CD38 expression on Molt-4 cells edited with several exemplary CD38 gRNAs described herein. These results demonstrate a reduction in CD38 protein detected in cells edited using the CD38 gRNAs.
  • Second-generation CARs are constructed to target CD38.
  • An exemplary CAR construct consists of an extracellular scFv antigen-binding domain, using CD8 ⁇ signal peptide, CD8 ⁇ hinge and transmembrane regions, the 4- IBB costimulatory domain, and the CD3 ⁇ signaling domain.
  • the anti-CD38 scFv sequence may be obtained from any anti-CD38 antibody known in the art, such those referenced herein.
  • CAR cDNA sequences for the target are sub-cloned into the multiple cloning site of the pCDH-EF1 ⁇ c-MCS-T2A-GFP expression vector, and lentivirus is generated following the manufacturer's protocol (System Biosciences).
  • Lentivirus can be generated by transient transfection of 293TN cells (System Biosciences) using Lipofectamine 3000 (ThermoFisher).
  • the exemplary CAR construct is generated by cloning the light and heavy chain of an anti-CD38 antibody, to the CD8 ⁇ hinge domain, the ICOS transmembrane domain, the ICOS signaling domain, the 4- IBB signaling domain and the CD3 ⁇ , signaling domain into the lentiviral plasmid pHIV-Zsgreen.
  • Human primary T cells are isolated from Leuko Pak (Stem Cell Technologies) by magnetic bead separation using anti-CD4 and anti-CD8 microbeads according to the manufacturer's protocol (Stem Cell Technologies). Purified CD4+ and CD8+ T cells are mixed 1:1 and activated using anti-CD3/CD28 coupled Dynabeads (Thermo Fisher) at a 1:1 bead to cell ratio.
  • T cell culture media used is CTS Optimizer T cell expansion media supplemented with immune cell serum replacement, L-Glutamine and GlutaMAX (all purchased from Thermo Fisher) and 100 lU/mL of IL-2 (Peprotech).
  • T cell transduction is performed 24 hours post activation by spinoculation in the presence of polybrene (Sigma).
  • CAR-T cells are cultured for 9 days prior to cryopreservation. Prior to all experiments, T cells are thawed and rested at 37°C for 4-6 hours.
  • the cytotoxicity of target cells is measured by comparing survival of target cells relative to the survival of negative control cells.
  • CD38 cytotoxicity assays wildtype and CRISPR/Cas9 edited cells of a CD38-expressing cell line, such as MOLT-4, are used as target cells. Wildtype Raji cell lines (ATCC) are used as negative controls for both experiments.
  • CD34+ cells may be used as target cells, and CD34+ cells deficient in CD38 or having reduced expression of CD38 may be generated as described in Example 1.
  • Target cells and negative control cells are stained with CellTrace Violet (CTV) and CFSE (Thermo Fisher), respectively, according to the manufacturer's instructions. After staining, target cells and negative control cells are mixed at 1:1.
  • CTV CellTrace Violet
  • CFSE Thermo Fisher
  • Anti-CD38 CAR-T cells were used as effector T cells.
  • Non-transduced T cells (mock CAR-T) are used as control.
  • the effector T cells are co-cultured with the target cell/negative control cell mixture at a 1:1 effector to target ratio in duplicate.
  • a group of target cell/negative control cell mixture alone without effector T cells is included as control.
  • Cells are incubated at 37°C for 24 hours before flow cytometric analysis. Propidium iodide (ThermoFisher) is used as a viability dye.
  • the fraction of live target cell to live negative control cell (termed target fraction) is used.
  • Specific cell lysis is calculated as ((target fraction without effector cells - target fraction with effector cells)/(target fraction without effectors)) x 100%.
  • Example 4 Effect of anti-CD38 antibody drug conjugates on Engineered HSCs
  • Genetically modified cells produced using the gRNAs shown in Tables 1 and 2 may be evaluated for killing by antibody-drug conjugates, such as belantamab mafodotin.
  • Frozen CD34+ HSPCs derived from mobilized peripheral blood are thawed and cultured for 72 h before electroporation with ribonucleoprotein comprising Cas9 and an sgRNA. Samples are electroporated with the following conditions: i.) Mock (Cas9 only), ii. KO sgRNA (such as any one of the CD38 gRNAs shown in Tables 1-5)
  • the percentage of CD38-positive cells is assessed by flow cytometry, confirming that editing with the CD38 gRNAs is effective in knocking out CD38.
  • the editing events in the HSCs result in a variety of indel sequences.
  • CD34+ HSPCs are edited with 50% of standard nuclease (e.g., Cas9, Cpf1) to gRNA ratios. The bulk population of cells are analyzed prior to and after treatment with the antibody-drug conjugate. Following treatment with the antibody-drug conjugate, CD38-modified cells are enriched so that the percentage of CD38 deficient cells increased. (iii) In vitro differentiation of CD34+ HSPCs
  • gRNAs (Synthego) were designed as described in Example 1.
  • mPB CD34+ HSPCs are purchased from Fred Hutchinson Cancer Center and thawed according to manufacturer's instructions. These cells are then edited via CRISPR/Cas9 as described in Example 1 using the CD38-targeting gRNAs described herein, as well as a non-CD38 targeting control gRNA (gCtrl) that is designed not to target any region in the human or mouse genomes.
  • LT-HSCs long term-HSCs
  • the percentages of LT-HSCs following editing with the specified CD38 gRNAs is assessed.
  • This assay may be performed, for example, at the time of cry opreservation of the edited cells, prior to injection into mice for investigation of persistence of CD38KO cells in vivo.
  • the edited cells are cryopreserved in CryoStor® CS10 media (Stem Cell Technology) at 5x10 6 cells/mL, in a 1 mL volume of media per vial. Investigating engraftment efficiency and persistence of CD38KO mPB CD34+ HSPCs in vivo
  • mice Female NSG mice (JAX) that are 6 to 8 weeks of age, are allowed to acclimate for 2-7 days. Following acclimation, mice are irradiated using 175 cGy whole body irradiation by X-ray irradiator. This was regarded as day 0 of the investigation. At 4-10 hours, following irradiation, the mice are engrafted with the CD38KO cells generated during any of the CD38 gRNAs described herein or control cells edited with gCtrl. The cryopreserved cells are thawed and counted using a BioRad TC-20 automated cell counter.
  • the number of viable cells is quantified in the thawed vials, which is used to prepare the total number of cells for engraftment in the mice.
  • Mice are given a single intravenous injection of 1x10 6 edited cells in a 100 ⁇ L volume. Body weight and clinical observations are recorded once weekly for each mouse in the four groups.
  • mice are sacrificed, and blood, spleens, and bone marrow are collected for analysis by flow cytometry. Bone marrow is isolated from the femur and the tibia. Bone marrow from the femur is also used for on-target editing analysis. Flow cytometry is performed using the FACSCantoTM 10 color and BDFACSDivaTM software.
  • Cells are generally first sorted by viability using the 7AAD viability dye (live/dead analysis), then Live cells are gated by expression of human CD45 (hCD45) but not mouse CD45 (mCD45). The cells that are hCD45+ are then further gated for the expression of human CD 19 (hCD19) (lymphoid cells, specifically B cells). Cells expressing human CD45 (hCD45) were also gated and analyzed for the presence of for various cellular markers of the myeloid lineage.
  • mice engrafted with the CD38KO cells are expected to have significantly lower levels of hCD38+ cells compared to the mice engrafted with control cells at weeks 8, 12, and 16.
  • the percentages of particular populations of differentiated cells, such as CD19+ lymphoid cells,, hCD14+ monocytes, and hCD11b+ granulocytes/neutrophils in the blood are quantified at weeks 8, 12, and 16 following engraftment in the mice engrafted with CD38KO cells or control cells.
  • the levels of hCD19+ cells, hCD14+ cells, and hCD11b+ cells in the blood were equivalent between the control and CD38KO groups, and the levels of these cells remained equivalent from weeks 8 to 16 post-engraftment. Comparable levels of hCD19+, hCD14+, and hCD11b+ cells in the blood indicate that similar levels of human myeloid and lymphoid cell populations were present in mice that received the CD38KO cells and mice that received the control cells.
  • amplicon- seq may be performed on bone marrow samples isolated at week 16 post-engraftment to analyze the on-target CD38 editing in mice that are engrafted with the edited CD38KO cells.
  • the percentages of hCD45+ cells and the percentage of hCD38+ cells are also quantified in the spleen of mice that are engrafted with control cells or CD38KO cells. Comparable levels of hCD45+ cells and reduced levels of hCD38+ cells between the groups of mice (engrafted with control cells or CD38KO cells) indicate the long-term persistence of CD38KO HSCs in the spleens of NSG mice.
  • the percentages of hCD14+ monocytes, hCD11b+ granulocytes/neutrophils, CD19+ lymphoid cells, and hCD3+ T cells in the spleen are quantified. Comparable levels of hCD14+ cells, hCD11b+ cells, hCD19+ cells, and hCD3+ in the spleen between the control and CD38KO groups indicate that the edited CD38KO cells are capable of multilineage human hematopoietic cell reconstitution in the spleen of the NSG mice.
  • the percentage of hCD11b+ cells are quantified in the blood and the bone marrow of mice engrafted with control cells or CD38KO cells. Comparable levels of CD11b+ neutrophil populations observed in the mice engrafted with control cells and the CD38KO cells in both the blood and the bone marrow of the NSG mice indicates successful engraftment and differentiation.
  • the percentage of hCD123+ cells in the blood and the percentage of hCD123+ cells in the bone marrow, and the percentage of hCD10+ cells in the bone marrow are quantified in mice engrafted with control cells or CD38KO cells. Comparable levels of myeloid and lymphoid progenitor cells between the control and CD38KO groups indicated successful engraftment and development.
  • CD34+ cells from three different human donors were gathered and electroporated with ribonucleoprotein complexes containing Cas9 and an exemplary CD38 gRNAs (e.g., gRNA CD38-8, gRNA CD38-11, or gRNA CD38-7).
  • exemplary CD38 gRNAs e.g., gRNA CD38-8, gRNA CD38-11, or gRNA CD38-7.
  • the percent positive CD38+ cells, the CD38 geometric mean fluorescence intensity (gMFI), and the percent mock were determined (FIGURES 7A- 7C).
  • Percent mock was calculated by dividing a CD38-edited sample's gMFI by a mock electroporated control gMFI. The results show that at 5 days post-electroporation all three donor's CD34+ cells showed an approximately 80% decrease in CD38 surface protein expression. These results demonstrated the effectiveness of the CD38 gRNAs of the disclosure at dramatically decreasing CD38 expression, for example at 5 days post electroporation in cells from multiple different human donors.
  • Table 10 INDEL Spectrum Data from CD38 Editing Using Selected gRNAs on Donor HSPCs
  • CD38-editing by CRISPR directed by the three selected CD38-targeting gRNAs was evaluated in five different human donor CD34+ cell samples: the three donor samples from FIGURES 7A-8B and two additional different human donors.
  • CD38 editing efficiency in the HSPCs was evaluated at 2, 5, and 7 days post electroporation (FilGURE 10A) by TIDE/ICE, and the percent CD38+ cells in the CD34+ cell samples were determined at 2, 5, 7, and 9 days post electroporation (FIGURE 10B). Data represent the average of data from all five donor samples. The results showed that CD38 editing efficiency persists and remains consistent at 2, 5, and 7 days post-electroporation. Concordantly, the percent CD38+ cells showed an approximately 80% decrease at 5 days post electroporation that persists at least to 9 days post electroporation. These results demonstrated that CD38 editing using the CD38 gRNAs is stable, persisting at least a week after electroporation, and that surface CD38+ protein expression is similarly stable after surface expression catches up to the gene editing.
  • Example 7 Evaluating CD38 Editing and Growlh/Viabilily of Edited THP-1 Cells [00191] The effects of CD38-editing using the selected CD38 gRNAs of Example 6 were examined in THP-1 cells.
  • THP-1 cells are human monocytic cells derived from an acute monocytic leukemia patient. Evaluating the effects of CD38-editing in such a proliferative cell line may better detect any alteration in growth or viability of edited cells and provides a further test of the effectiveness of CRISPR induced CD38 gene modification using the gRNAs of the disclosure.
  • THP-1 cells were electroporated at day 0 with ribonucleoprotein complexes comprising Cas9 and one of the exemplary CD38 gRNAs (gRNA CD38-8, gRNA CD38-11, or gRNA CD38-7). The total cell count and the percentage of cells that were viable cell were determined daily for 12 days post-electroporation (FIGURES 11A-11B). Edited samples were compared to "wild-type" unedited THP-1 cells. The results show that CD38- edited THP-1 cells proliferated over the 12 day test period, with percent viable cell levels rising up to match wildtype THP-1 cells by 5 days post-electroporation. These results show that editing of CD38 in the THP-1 cells conveys no advantage or disadvantage in regards to growth or viability of cells, suggesting that editing of CD38 did not impact growth or viability.
  • CD38 editing efficiency, CD38 RNA expression levels, and percent of THP-1 cells that were positive for CD38 surface protein were determined to evaluate editing using the CD38 gRNAs in THP-1 cells (FIGURES 12A-12C).
  • CD38 editing efficiency and transcript expression were determined by DNA sequencing and RNA quantification, respectively.
  • the percentage of CD38+ cells was determined by FACS.
  • the results showed that the CD38 gRNAs directed CRIS PR-induced CD38 editing with high efficiency, producing an approximately 80% decrease in CD38-encoding RNA transcripts and 71-91% decrease in the percentage of CD38+ cells.
  • the results showed that the CD38 gene edits, transcript decrease, and surface protein decreases persist to at least 11 days post- electroporation.
  • HSCs and HSPCs can be detected and their capacity for growth and division evaluated by an in vitro colony forming cell assay.
  • CD34+ HSPCs were isolated from a human donor and electroporated with ribonucleoprotein complexes comprising Cas9 and CD38 gRNAs described herein.
  • the colony forming capacity of the CD38-edited HSPCs was evaluated using a STEMvisionTM device following the manufacturer's protocol, with mock electroporated HSPCs as control. 400 cells were plated in duplicate.
  • BFU-E protocol measured erythroid differentiated cell colonies
  • G/M/GM protocol measured myeloid differentiated cell colonies
  • GEMM measured colonies of a mixture of differentiated cells
  • INDEL spectrum was evaluated for human donor HSPCs in CD38 edited cells.
  • HSPCs were electroporated with ribonucleoprotein complexes comprising Cas9 and CD38 gRNAs described herein.
  • INDEL analysis was performed using TIDE/ICE on the bulk HSPCs in culture 2 days after electroporation and compared to INDELs of colony forming HSPCs assessed 14 days after electroporation (FIGURES 14A-14C). The results showed that the INDEL patterns for editing with a given CD38-specific gRNA persist at least 14 days after electroporation and the INDEL patterns of edited HSPCs that formed colonies are similar to the patterns of bulk HSPCs in culture.
  • Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context.
  • the disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
  • any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods described herein, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

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